AU2021246472B2 - Sterilization of self-assembling peptides by irradiation - Google Patents

Abstract

Gamma ray and e-beam irradiation provided efficient sterilization of certain self- assembling peptides (including RADA16 in solution) without substantial degradation of the major peptide, while, e.g., another self-assembly peptide, QLEL12 was significantly degraded following irradiation. Irradiation sterilization enhances the rheological property of, for example, RADA16 hydrogel once applied to tissue at a physiological pH. The rheological property increase can result in higher efficacy in a variety of biomedical applications.

Description

STERILIZATION OF SELF-ASSEMBLING PEPTIDES BY IRRADIATION

Priority

This application claims priority to United States provisional Application No.

63/002,882, filed on March 31, 2020.

Field of Invention

This invention relates to sterilization of certain medical gels, more specifically,

gels containing so-called self-assembling peptides.

Background of the Invention

Self-assembling peptides (sometimes abbreviated as "SAPs") are a type

of peptides which assemble spontaneously into highly organized nanostructures when

placed in aqueous environment and a chemical or physical change in surrounding

conditions occur. One other well-known structure is a nanofibrous biopolymer structure

formed by natural collagen. A class of SAPs relevant for this invention consists of

alternating hydrophilic and hydrophobic amino acid residues capable of forming beta

sheets. They autonomously assemble into well-ordered nanostructures in neutral water,

while they can temporarily disassemble into individual molecules when high shearing

force is applied to them. SAPs can form a hydrogel (also known as SAP gels)

depending on their environment such as pH and/or osmolality; for example, they are

capable of forming a hydrogel, when they are placed in the body at near neutral pH,.

SAP gels have been previously described as being used for a variety of medical

applications, e.g., improved wound healing, inducement of homeostasis; reduction of

adhesion in interior tissues, particularly, in context of surgery; as temporary tissue-void matrix fillers, facilitating ingrowth of natural tissue into such a void. Particular SAPs are described in US Patents Nos. 5,670,483; 5,955,343; 9,724,448; 10,596, 225 and Int'l

Pat. AppIn. Pub. W02014/136081; and foreign equivalents thereof.

Sterilization is a very important step in the manufacturing process for most

biomaterials, including for self-assembling peptide solutions. PuraStat©(RADA16 = Ac

RADARADARADARADA-NH2 = SEQ ID NO:1; about 2.5%) is customarily filtered for

sterilization (see, e.g., Int'l. Pat. AppIn. Pub. No. WO 2014/008400); however,

PuraStat©'s viscosity at higher concentrations is the main obstacle to its filtration, which

in addition to losses in the tubing, results in substantial peptide losses. In this method

for sterilizing, a solution of SAPs is forced through a porous filter, wherein the sterilizing

filter has an average pore size of 0.22 pm (US Pat. AppIn. No. 2015/019735). As

another method, some thermally stable self-assembling peptide solutions can be

sterilized by autoclaving treatment at about 121 °C for about 25 minutes (US Pat. AppIn.

No. 10/369,237).

Furthermore, an additional ethylene oxide sterilization step is required for the

outer part of PuraStat© products. And, it was discovered that autoclaving cannot be

used for some SAPs, such as RADA16 in solution, because of its complete thermal

degradation (US Pat. AppIn. Pub. No. 2017/0202986). Thus, another new sterilization

method has been needed to reduce losses, particularly, for RADA16 and other SAPs.

Gamma irradiation sterilization of self-assembling peptides including RADA16

was described in passing in a prior publication (US Pat. AppIn. Pub. No. 2016/0317607;

at paragraph [0052]); but no specific conditions or actual effect of irradiation on the

structure and properties of self-assembling peptides, including RADA16, have been described so far. In fact, peptide structure can be changed by the reactive radical species generated by irradiation such as gamma ray, X-ray, and e-beam. Such a structural change can be governed by multiple factors including the amino acid composition, reactive residue position and possibly the conformation acquired by each macromolecule (Vieira R et al, Biol. Pharm. Bull. 2013, 36(4) 664-675). For example, regardless of the different sequences of the peptides in that article, all the tested nine peptides showed a progressive degradation by gamma ray irradiation up to 15 kGy.

Considering that even higher doses of irradiation than 15 kGy may be required for

sterilization process, numerous peptides cannot be sterilized with gamma ray irradiation

without substantial degradation. It is well known that the side chains of aromatic amino

acids (i.e., Phenylalanine (F), Tyrosine (Y), Tryptophan (W), Histidine (H), and Proline

(P)) and sulfur-containing amino acids (i.e., Cysteine (C)) are especially weak to attack

by reactive radical species (Annu. Rev. Biochem., 62, 797-821 (1993) and Vieira R et al,

Biol. Pharm. Bull. 2013, 36(4) 664-675). By way of background, the followings SAPs,

RADA16 (Ac-RADARADARADARADA-NH2 (SEQ ID NO:1)), KLD12 (Ac

KLDLKLDLKLDL-NH2 (SEQ ID NO:2)), and IEIK13 (Ac-IEIKIEIKIEIKI-NH2 (SEQ ID

NO:3)), do not include aromatic amino acids or sulfur-containing amino acids.

On the other hand, irradiation sterilization can also affect the secondary structure

of self-assembling peptides and their fibrous structure. As mentioned, RADA16, KLD12

and IEIK13 have beta-sheet conformation, and these molecules self-assemble to form

an ordered nanofibrous structure. Irradiation sterilization process may potentially

change the secondary structure and the nanofiber structure of peptide, which could then

result in the undesirable change of its rheological properties.

Thus, there exists a need for new sterilization methods that work advantageously

with self-assembling peptides. For the reasons cited above, sterilization by irradiation

has been avoided so far.

Any discussion of the prior art throughout the specification should in no way be

considered as an admission that such prior art is widely known or forms part of the

common general knowledge in the field.

Unless the context clearly requires otherwise, throughout the description and the

claims, the words "comprise", "comprising", and the like are to be construed in an

inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the

sense of "including, but not limited to".

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Appearance of PuraStat©(RADA16 2.5%) and IEIK13 1.3% before

(Figure 1A) and after gamma-irradiation at 28 kGy (Figure 1B).

Figure 2. Mass spectra of PuraStat©(RADA16 2.5%) (A) before gamma

irradiation, (B) after gamma irradiation at 40 kGy, and (C) after autoclaving at 121 °C for

20 min.

Figure 3. Mass spectra of PuraStat© (RADA16 2.5%) (A) after X-ray irradiation at

25 kGy and (B) after X-ray irradiation at 40 kGy.

Figure 4. Mass spectra of PuraStat© (RADA16 2.5%) (A) after e-beam irradiation

at 25 kGy and (B) after e-beam irradiation at 40 kGy.

Figure 5. Mass spectra ofIEIK13 1.3% (A) before irradiation; (B) after gamma

irradiation at 40 kGy; (C) after X-ray irradiation at 25 kGy; (D) after X-ray irradiation at

40 kGy; (E) after e-beam irradiation at 25 kGy; and (E) after e-beam irradiation at 40

kGy.

Figure 6. Mass spectra of QLEL12 0.15% (A) before irradiation; (B) after gamma

irradiation at 23 kGy; (C) after X-ray irradiation at 25 kGy, (D) after X-ray irradiation at

40 kGy, (E) after e-beam irradiation at 25 kGy, and (F) after e-beam irradiation at 40

kGy.

Figure 7. Frequency test of PuraStat©(RADA16 2.5%) with gamma irradiation at

0.1% of strain with 40 mm cone-plate (N=3, bars represent SD).

Figure 8. Frequency test of PuraStat©(RADA16 2.5%) with gamma irradiation

after gelation at 0.1% of strain with 40 mm cone-plate. Samples were treated with

DMEM for 20 min (N=3, bars represent SD).

Figure 9. Thixotropic test of PuraStat©(RADA16 2.5%) with gamma irradiation at

1 Hz of frequency and 0.1% of strain with 40 mm cone-plate. Initial storage modulus (G')

was measured for 1 min before 1st shearing at 1000 s 1. After the 1st shearing for 1

minute, G' recovery was recorded for 1 hour to exhibit the thixotropic behavior of

PuraStat©. This test was duplicated.

SUMMARY OF THE INVENTION

In a first aspect, the present disclosure provides a method of sterilizing a self

assembling peptide (SAP) solution, the method comprising:

(a) placing one or more containers with the solution of self-assembling peptide

into an irradiation machine, said self-assembling peptide capable of

forming a hydrogel when applied to a biological tissue at about neutral pH;

and

(b) exposing the container to gamma ray, X-ray and/or e-beam irradiation at a

predetermined dose so that the self-assembling peptide solution is

sterilized to a pre-determined Sterility Assurance Level (SAL) without

substantial degradation of the peptide, wherein the concentration of the

degradation products of the full-length peptide ("major peptide") in the

solution post-irradiation ranges from 0.1% to 20% by weight while its

desired physical properties are maintained substantially at the same level

or improved;

wherein the peptide is selected from the group consisting of RADA16, KLD12,

and IEIK13; and

wherein the desired physical properties are storage modulus and/or viscosity.

In a second aspect, the present disclosure provides a sterilized solution of

self-assembling peptide made by the method of the first aspect.

In a third aspect, the present disclosure provides a method of using the

sterilized solution of the second aspect, comprising applying the solution to a

biological tissue.

In a fourth aspect, the present disclosure provides use of a sterilized solution

of the self-assembling peptide for treating or preventing a disease or condition,

wherein the sterilized solution is obtained by the method of the first aspect.

In the present disclosure, the effect of sterilization by irradiation on self

assembling peptide solutions was evaluated, specifically, with the following peptides:

PuraStat©(Ac-RADARADARADARADA-NH2, RADA16 (SEQ ID NO:1)), IEIK13 (Ac

IEIKIEIKIEIKI-NH2 (SEQ ID NO:3)), and QLEL12 (Ac-QLELQLELQLEL-NH2 (SEQ ID

NO:4)). It was unexpectedly found that gamma-irradiation sterilization enhanced the

rheological properties of certain self-assembling peptide solutions and hydrogels

without the anticipated noteworthy degradation, while certain other peptides showed

the expected significant degradation and viscosity drop after gamma irradiation (i.e.,

unlike RADA16 and IEIK13), while, e.g., another self-assembly peptide, QLEL12

(Ac-QLELQLELQLEL-NH2 (SEQ ID NO:4)) was significantly degraded following

irradiation. This invention is further based on the prophetic finding that the same

result can be expected with other similar irradiation sterilization methods including,

specifically, X-ray and e-beam, at least for the respective peptides. Thus, in some

embodiments, the method of sterilizing a self-assembling peptide solution comprises:

a) placing one or more containers with a solution of self-assembling peptide

into an irradiation machine, said self-assembling peptide capable of

forming a hydrogel when applied to a biological tissue at about neutral pH;

and

b) exposing the container to gamma ray, X-ray and/or e-beam irradiation at a

predetermined dose so that the peptide solution is sterilized without substantial degradation of the peptide while its desired biological and/or rheological property(ies) is/are maintained at the same level or improved.

In some embodiments, the peptides are selected from the group consisting of

RADA16, KLD12, and IEIK13. In related embodiments, such peptides are exposed to

the dose of 15 - 50 kGy, preferably 15 - 40 kGy, more preferably, to a minimum dose

resulting in a desired sterility assurance level (SAL), without substantial degradation

and/or substantial negative change in biological properties of these peptides. In some

embodiments, the peptide solution is irradiated by gamma-rays, X-rays, or e-beam. In

some embodiments, the over-all degradation of the total peptides in solution after

irradiation does not exceed 20%, more preferably, 10%, most preferably 5%, of the

amount of peptides prior to irradiation. In some embodiments, the desired biological or

physical property(ies) is/are selected from the group consisting of: hemostatic, anti

adhesion, prevention of re-bleeding, anti-stenosis, tissue occlusion, storage modulus

(e.g., in some embodiments, the storage modulus of the gelled solution is increased at

least by 10%, at least by 15% or at least by 20% post-irradiation), and viscosity, and

tissue void filling property, which are maintained within acceptable or improved

parameters after irradiation. In some embodiments, irradiation dose achieves sterility

assurance level (SAL) of at least 10-5, preferably 10-6, or less. In other embodiments,

using the bioburden testing described below in the example, the acceptable level of

contamination of the peptide solution pre-irradiation is 1000, 500,100, 15, 10, 9, 5, 2,

1.5, 1 CFU, or less. In some embodiments, the concentration of the degradation

products of the intact ("major" or "full-length") peptide in the solution post-irradiation

ranges from 0.1% to 5%. In some embodiments, the pH of the peptide solution post irradiation ranges from about 1.8 to 3.5. In some embodiments, the solution container is a plastic syringe, with or without an adapter nozzle. In such embodiments, care is taken to ensure that the plastic and rubber parts of the packaging also maintain their desired physical properties. While some yellowing of the plastic syringes may be expected and is normal, any rubberized material must preserve its plastic properties at an acceptable level.

In some embodiments, the gelled solution is further subjected to shearing to

reduce or restore its storage modulus.

Thus, the invention provides a sterilization method for self-assembling peptides.

According to the methods of the invention, in some embodiments, the solution is further

applied to a biological tissue, for example, during surgery, or after trauma involving

bleeding.

DETAILED DESCRIPTION OF THE INVENTION

As stated above, in the present disclosure, the effect of sterilization by

irradiation on self-assembling peptide solutions was evaluated, specifically, with the

following peptides: PuraStat©(Ac-RADARADARADARADA-NH2 (SEQ ID NO:1));

RADA16), IEIK13 (Ac-IEIKIEIKIEIKI-NH2(SEQ ID NO:3)), and QLEL12 (Ac

QLELQLELQLEL-NH2 (SEQ ID NO:4)). In some embodiments, the method of sterilizing

a self-assembling peptide solution comprises:

a) placing one or more containers with a solution of self-assembling peptide into

an irradiation machine, said self-assembling peptide capable of forming a

hydrogel when applied to a biological tissue (e.g., in situ) at about neutral pH;

and

b) exposing the container to gamma ray, X-ray and/or e-beam irradiation at a

predetermined dose so that the peptide solution is sterilized to a pre

determined Sterility Assurance Level (SAL) without substantial degradation of

the peptide while its desired biological and/or physical property(ies) is/are

maintained substantially at the same level or improved.

It was unexpectedly found that gamma-irradiation sterilization enhanced the

rheological properties of certain self-assembling peptide solutions and hydrogels without

causing any noteworthy degradation, while certain other peptides showed significant

degradation and viscosity drop after gamma irradiation, i.e., unlike RADA16 and IEIK13,

another self-assembling peptide, QLEL12 (Ac-QLELQLELQLEL-NH2 (SEQ ID NO:4))

was significantly degraded following irradiation. This invention is further based on the

prophetic finding that the same result can be expected with other similar irradiation sterilization methods including, specifically, X-ray and e-beam, at least for the respective peptides. In preferred embodiments, the composition and methods of the invention maintain or improve the desired biological property(ies) such as hemostatic, anti-adhesion, prevention of re-bleeding, anti-stenosis, tissue occlusion, storage modulus, viscosity, and tissue void filling property, etc. For example, in some embodiments, the storage modulus is increased by at least 5%, 10%, 15%, 20%, or more. If such increase is undesirable for certain applications, the gel can be thinned further by dilution or shearing by methods known in the art, turning it into the solution or otherwise reducing its storage modulus. In certain embodiments, the irradiated solution of SAPs remains clear and viscous.

In some embodiments, the SAPs comprise a sequence of amino acid residues

conforming to one or more of Formulas I-IV:

((Xaaneu-Xaa+)x(Xaaneu-Xaa-)y)n (I)

((Xaaneu-Xaa-)x(Xaane-Xaa+)y)n (II)

((Xaa+-Xaaneu)x(Xaa--Xaane)y)n (Ill)

((Xaa--Xaane)x(Xaa+-Xaane)y)n (IV)

Xaane represents an amino acid residue having a neutral charge; Xaa+ represents an

amino acid residue having a positive charge; Xaa- represents an amino acid residue

having a negative charge; x and y are integers having a value of 1, 2, 3, or 4,

independently; and n is an integer having a value of 1-5.

In some embodiments, the SAPs further comprise an amino acid sequence that

interacts with the extracellular matrix, wherein the amino acid sequence anchors the

SAPs to the extracellular matrix.

In some embodiments, the amino acid residues in the SAPs can be naturally

occurring or non-naturally occurring amino acid residues. Naturally occurring amino

acids can include amino acid residues encoded by the standard genetic code as well as

non-standard amino acids (e.g., amino acids having the D-configuration instead of the

L-configuration), as well as those amino acids that can be formed by modifications of

standard amino acids (e.g., pyrrolysine or selenocysteine). Suitable non-naturally

occurring amino acids include, but are not limited to, D-alloisoleucine(2R,3S)-2-amino

3-methylpentanoic acid, L-cyclopentyl glycine (S)-2-amino-2-cyclopentyl acetic acid.

In other embodiments, another class of materials that can self-assemble are

peptidomimetics. Peptidomimetics, as used herein, refers to molecules which mimic

peptide structure. Peptidomimetics have general features analogous to their parent

structures, polypeptides, such as amphiphilicity. Examples of such peptidomimetic

materials are described in Moore et al., Chem. Rev. 101(12), 3893-4012 (2001). The

peptidomimetic materials can be classified into four categories: a-peptides, p-peptides,

y-peptides, and 5-peptides. Copolymers of these peptides can also be used. Examples

of a-peptide peptidomimetics include, but are not limited to, N,N'-linked oligoureas,

oligopyrrolinones, oxazolidin-2-ones, azatides and azapeptides. Examples of p-peptides

include, but are not limited to, p-peptide foldamers, a-aminoxy acids, sulfur-containing

P-peptide analogues, and hydrazino peptides. Examples of y-peptides include, but are not limited to, y-peptide foldamers, oligoureas, oligocarbamates, and phosphodiesters.

Examples of 5-peptides include, but are not limited to, alkene-based 5-amino acids and

carbopeptoids, such as pyranose-based carbopeptoids and furanose-based

carbopeptoids.

In certain embodiments, the SAP is AC5@, AC5-V@, AC5-GT M or TK45, also

known as AC1, made by Arch Therapeutics, Inc. (see www.archtherapeutics.com).

In some embodiments, the SAP solution is contained in "storage and/or drug

delivery system", such as, for example, storage and/or delivery systems suitable for

peptide compositions described herein, for example, vials, bottles, beakers, bags,

syringes, ampules, cartridges, reservoirs, or LYO-JECTS©. Storage and/or delivery

systems need not be one in the same and can be separate. In specific embodiments,

SAPs is provided in plastic syringe, containing, about 10 ml, about 7.5 ml, about 5,

about 2.5., about 1, or about 0.5 ml of a SAP solution. In certain embodiments, the

plastics (e.g., plastic syringe) may acquire a yellowish tint after irradiation, which is

normal, and does not affect biomedical characteristics of the therein contained SAP

solution.

In some embodiments, such storage and delivery system may further contain a

"nozzle" which refers to a generally thin, cylindrical object, often with a narrow end and

a wide end, which is adapted for fixing onto a delivery device described herein. In some

embodiments, the terms "nozzle" and "cannula" are used interchangeably. Nozzles are

composed of two connection points or ends, a first connection point or end to connect to

a delivery system (e.g., a syringe) and a second connection point which may serve as

the point where delivery of pharmaceutical composition is administered or as a point to

connect to a secondary device (e.g., a catheter).

Thus, the invention provides methods of making sterilized solutions of the self

assembling peptides. The invention also provides use of such sterilized solutions

applied to a biological tissue, e.g., in situ, for example, during surgery or after trauma involving bleeding, or use of so-sterilized solutions in treatment or prevention of other diseases or conditions; for example, as described in Int'l Pat. AppIn. W02014/133027, or as described in US Pat. AppIn. Pub. Nos. 2011/02101541 and occlude site of tissue damage; or as described US Pat. AppIn. Pub. No. 2016/0287744 for vascular embolization; or as described in US Pat. AppIn. No. 16/085,803 for occlusion of cerebrospinal fluid leakage, or as described in Int'l Pat. Pub. AppIn. No.

W02013/133414 as mucosa elevating agent; or as described in Int'l Pat. Pub. AppIn.

No. W02014/141160 for bile leakage occlusion; or as described in US Pat. AppIn. No.

16/885,753 for anti-adhesion of tissues; or as described in US Pat. AppIn. No.

16/085,804 for pancreatic fistula occlusion; or as described in US Pat. AppIn. No.

15/124,639 for bronchial obstruction; or as described in Int'l Pat. AppIn. Pub. No.

W02015/138,478 for treating pulmonary bulla collapse; or as described in Int'l Pat.

AppIn. Pub. No. W02015/019,738 for treatment of pulmonary leakage; or as described

in Int'l Pat. AppIn. Pub. No. W02015/196020 for filling dental bone voids; or as

described US Pat. AppIn. Pub. No. 2017/0128622 for filling bone voids; or as described

in US Pat. AppIn. No. 16/312,878 for the prevention of esophageal structure after

endoscopic dissection, and foreign equivalents of any of the aforementioned

publications, and other methods known in the art. Accordingly, in some embodiments,

the invention provides the use of a sterilized solution of the self-assembling peptide for

treating or preventing aforementioned disease or condition, wherein the sterilized

solution is obtained by the methods of the invention. In certain such embodiments, the

self-assembling peptide solution exhibits a post-irradiation mass spectrometric (MS)

profile substantially as shown in corresponding post-irradiation profiles of FIGS. 2-6 and/or as described in the Examples. For example, in the case of PuraStat©, the additional major Mz peaks are observed at 836/1670, 1100, and 1513 m/z.

An average bioburden <1,000 CFU is the typical sterility of PuraStat© before

sterilization. In this case, to achieve a sterility assurance level, SAL, of 10-6, the range of

irradiation dose should be between 25 kGy and 40 kGy. With gamma and X-ray

methods, about up to 20% of the over-all peptides may degrade during sterilization

process. With e-beam method, about up to 10% of the over-all peptides may degrade

during sterilization process. PuraStat rheology increases after sterilization by gamma

ray, X-ray, which can alter its hemostatic efficacy positively or negatively. However,

PuraStat'rheology does not change after e-beam sterilization.

In certain embodiments, using the bioburden testing described below in the

Examples, the acceptable level of contamination of the peptide solution pre-irradiation

may be <1000, <500, <100, <15, <10, <9, <5, <2, <1.5, <1 CFU, or less per product unit.

The preferred bioburden pre-sterilization is < 9 CFU. Thus, under 9 CFU, the range of

irradiation dose can be selected between 15 kGy and 24 kGy. While the low end of the

irradiation dose range may be determined by the bioburden of the pre-irradiated product,

the high end of the range is chosen based on the configuration and choice of the

irradiator machine and set at a minimum required to achieve desirable sterility

assurance level (SAL).

With gamma and X-ray methods, up to 10% of the peptides may degrade during

sterilization process. With e-beam method, the peptides may not significantly degrade

during sterilization process. Even in this case, PuraStat rheology increases after

gamma-ray and X-ray sterilization, which may somewhat change its hemostatic efficacy positively or negatively. Thus, PuraStat rheology does not change after e-beam sterilization and thus may be preferred, if no or minimal change in rheological properties and its hemostatic properties is desirable. Therefore, e-beam irradiation may be particularly preferred for PuraStat©.

In some embodiments, the concentration of degraded full-length peptide ("major

peptide") in the solution post-irradiation ranges from 0.1% to 5%, 0.1% to 4%, 0.1% to

3%, 0.1% to 2.5%, 0.1% to 2%, or 0.1% or 1.5% or less. In some embodiments,

RADA16, KLD12, or IEIK13 solution are irradiated with a total dose, which may depend

on its pre-irradiation bioburden, including, for example, 15-50 kGy, 25 kGy +/- 15 kGy,

40 kGy - 10 kGy, 35 kGy -10 kGy, 30 kGy -10 kGy, 15 kGy - 24 KGy, 25 kGy -10 kGy,

20 kGy -10 kGy, 10 kGy -15 kGy, and 12 kGy -14 kGy. Doses around 12-14 kGy appear

to be optimal for gamma irradiation, however, the peptide solution may also be

irradiated with similar doses with X-ray or e-beam.

In some embodiments, the "storage and/or drug delivery system", for example, a

plastic syringe contained in a blister pack, is irradiated in batches of at least: 100, 200, 300, 400,

500, 600, 700, 800, 900, 1000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000

units or more at a time. Generally, it is preferred that the total exposure of any sample in

an irradiated batch does not exceed 100 kGy, more preferably 60 kGy, most preferably,

50 kGy, or less as described herein.

In some embodiments, the major peptide's degradation (also referred to as "full

length peptide's degradation") after the irradiation does not exceed 20% of the amount

of the peptide prior to the irradiation and, preferably, does not exceed 18%, 16%, 14%,

12%, 10%, 8%, 5%, 3%, or 1%. In some embodiments, the total dose is achieved over a period of time sufficient to achieve necessary sterility of the solution. For example, the dose of 40 kGy can be delivered by radiation intensity 6.3 kGy/hr for about 6 hrs and 21 min. Other combination can be found in Example 1. In some embodiments, a constant dose was delivered over a number of hours, e.g., about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 hours.

In some embodiments, samples are irradiated with gamma ray doses, for example,

of about 23, about 25, about 28 or about 40 kGy, or other doses indicated above with an

instrument, for example, such as Gammacell 220© High Dose Rate Co-60 Irradiator in

VPTrad (www.vptrad.com; MDS Nordion, Ottawa, Canada).

In other embodiments, samples are irradiated with X-ray with doses of about 25

kGy to about 40 kGy, or the doses as indicated above, with a Mevex accelerator (with

the following settings: 10 MeV, 20 kW).

In general, X-ray frequency range is 3x10 16-3x10 19 Hz, while the frequency range

of gamma-rays frequency is 3x1019 or higher.

In other embodiments, samples are irradiated with e-beam at about 25 to about

40 kGy with a Mevex accelerator (with the following settings: 10 MeV, 20 kW) (see

mevex.com/linacs/1Omev-system-e-beam-sterilization-for-medical-devices/).

In some embodiments, the desired biological and physical property(ies) is/are

selected from the group consisting of: hemostatic, anti-adhesion, prevention of re

bleeding, anti-stenosis, tissue occlusion, storage modulus, viscosity, tissue void filling

property, mucosa elevation, and wound healing.

In preferred embodiments, the irradiation dose achieves sterility assurance level

(SAL) of at least 10-, 10-6, or less.

The invention also provide the sterilized solution of self-assembling peptide made

by the methods described herein. Such comprising applying the solution to a biological

tissue, for example, during surgery or after trauma involving bleeding, to a site with

substantially neutral pH, resulting in gelling. In some embodiments, such site is internal,

while in other embodiment the site can be external such surface sutures, cuts, or

scrapes.

Other aspects of the invention would be apparent to those of skill in the art based

on the present description, including the Examples and the appended claims.

Example 1: Irradiation conditions

Samples were irradiated with gamma rays at 23, 25, 28 and 40 kGy with VPTrad

Gammacell 220© High Dose Rate Co-60 Irradiator. In some embodiments, the run dose

rate and duration time for 40 kGy irradiation were 6.30 kGy/hr and 6 hours 20 minutes

58 seconds, respectively. In other embodiments, the run dose rate and duration time for

28 kGy irradiation were 4.40 kGy/hr and 6 hours 22 minutes 31 seconds, respectively.

In other embodiments, the run dose rate and duration time for 25 kGy irradiation were

6.58 kGy/hr and 3 hours 47 minutes 42 seconds, respectively. In other embodiments,

the run dose rate and duration time for 23 kGy irradiation were 6.58 kGy/hr and 3 hours

29 minutes 29 seconds, respectively.

In other embodiments, samples were irradiated with X-ray at 25 kGy and 40 kGy

with a Mevex accelerator (10 MeV, 20 kW).

In other embodiments, samples were irradiated with e-beam at 25 and 40 kGy

with a Mevex accelerator (10 MeV, 20 kW).

Example 2: HPLC conditions

HPLC tests were performed to evaluate the major peptide content after

irradiation tests. An Agilent HPLC 1100 (Agilent Technologies) was used for this study.

The column temperature was kept at 25 °C.

For RADA16 samples, solvent A was water with 0.1% TFA and solvent B was

80% acetonitrile with 0.1% TFA. Gradient of solvent B was controlled from 10% to 40%

in 20 min and 40% for another 5 min at 25 °C. Agilent Zorbax 300SB-C18 column (4.6

mm X 250 mm, 5tm, 300 A) was used for this test. PuraStat© (RADA16 2.5%) (40 mg)

was mixed with 10 pL of DH2O and the mixture were vortexed. The mixture was further

mixed with 500 pL of formic acid and vortexed. This mixture was then mixed with DH2O

(4,450 pL) and vortexed. 20 tL samples were injected using an Agilent autosampler.

For IEIK13 samples, solvent A was water with 0.1% TFA and solvent B was 90%

Acetonitrile with 0.1% TFA. Gradient of solvent B was controlled from 20% to 43% in 8

min, from 43% to 70% between 8 and 9.5 min, and from 70% to 95% between 9.5 and

30 min, and 95% for another 5 min at 25 0C. An Agilent PLRP-S column (4.6 mm X 250

mm, 8 tm, 300 A) was used for this test. A 1.3% solution of IEIK13 was diluted with

water with 0.1% TFA to 0.075% and the mixture was vortexed. 35 tL samples were

injected using an Agilent autosampler.

For QLEL12 samples, solvent A was water and solvent B was 80% acetonitrile.

Gradient of solvent B was controlled from 20% to 80% in 15 min and 80% for another 7 min at 25°C. An Agilent PLRP-S column (4.6 mm X 250 mm, 8tm, 300 A) was used for this test. 0.15% w/v of QLEL12 solution was diluted to 0.01% with DH2O and vortexed.

50 tL samples were injected using an Agilent autosampler.

Example 3: Mass spectrometry conditions

Mass spectrometry tests were carried out to investigate the degradation of the

peptides after irradiation sterilization. An Agilent LC/MSD ion trap mass spectrometer

was used for this study. The sample solutions were prepared as described above for the

HPLC samples. Each sample was injected at 9 pL/min with a syringe pump. Mass

spectrum was recorded for 1 minute.

Example 4: Rheological properties of irradiated self-assembling peptides

The rheological properties of samples were evaluated using a rheometer (DHR1,

TA Instruments) with 40 mm cone and plate. Peptide solution (700 pL) was placed on

the rheometer plate and excess solution was gently removed by a metal spatula.

Measurements were performed after 2 minutes of relaxation time at 37 °C.

Frequency tests were performed at 0.1% of strain from 0.1 Hz to 10 Hz. Frequency

tests after gelation were performed under the same conditions for 20 minutes after 3 mL

of DMEM was gently added around the cone and the plate.

Thixotropic tests were carried out with the following method. A shear rate of 1000

S-1 was applied for 1 minute to reset all rheological properties, then 1 Hz of frequency at

0.1% of strain was applied for 60 minutes to record thixotropic behaviors. This

sequence was repeated, and the thixotropic property were then analyzed.

Example 5: Appearance and pH

The appearance and pH of PuraStat after gamma irradiation are shown in

Figure 1 and Table 1 (N=3).

Table 1. Appearance and pH of PuraStat©(RADA16 2.5%) after gamma irradiation. Testing conditions Appearance pH

PuraStat© control Clear and viscous 2.2

PuraStat irradiated (gamma) at 23 kGy Clear and viscous 2.3

PuraStat irradiated (gamma) at 25 kGy Clear and viscous 2.3

PuraStat irradiated (gamma) at 28 kGy Clear and viscous 2.3

PuraStatOirradiated (gamma) at 40 kGy Clear and viscous 2.3

The appearance and pH of PuraStat'after gamma irradiation were only slightly altered.

The pH of the PuraStat© control was 2.2. The pH of PuraStat© after gamma irradiation at

23, 25, 28, and 40 kGy were 2.3.

The appearance and pH of IEIK13 1.3% after gamma irradiation are also shown

in Figure 1 and Table 2.

Table 2. Appearance and pH of1EIK13 1.3%, KLD12 1.3% and QLEL12 0.15% after gamma irradiation. (N=3) Testing conditions Appearance pH

1EIK13 control Clear and viscous 3.0

1EIK13 irradiated (gamma) at 28 kGy Clear and viscous 3.0

1EIK13 irradiated (gamma) at 40 kGy Clear and viscous 3.0

KLD12 control Clear and viscous 2.2

KLD12 irradiated (gamma) at 40 kGy Clear and viscous 2.2

QLEL12 control Clear and viscous 7.0

QLEL12 irradiated (gamma) at 23 kGy Clear and watery 6.7

QLEL12 irradiated (gamma) at 25 kGy Clear and watery 6.5

QLEL12 irradiated (gamma) at 40 kGy Clear and watery 5.9

The appearance and pH of1EIK13 (1.3%) after gamma irradiation were not

changed. The pH of 1EIK13 control was 3.0. The pH of1EIK13 (1.3%) after gamma

irradiation at 28 kGy and at 40 kGy was 3.0.

The appearance and pH of KLD12 (1.3%) and QLEL12 (0.15%) after gamma

irradiation were also shown in Table 2. The appearance and pH of KLD12 (1.3%) after

gamma irradiation were not changed. The pH of KLD12 control was 2.2. The pH of

KLD12 (1.3%) after gamma irradiation at 40 kGy were 2.2.

However, the appearance and pH of QLEL12 after gamma irradiation were

significantly altered. The pH of the QLEL12 control was 7.0. The pH of QLEL12 after

gamma irradiation at 23, 25 and 40 kGy were 6.7, 6.5 and 5.9, respectively, and the

samples became watery after gamma irradiation.

The appearance and pH of PuraStat after X-ray and e-beam irradiation are also

shown in Table 3 (N=3).

Table 3. Appearance and pH of PuraStat©(RADA16 2.5%) after X-ray and e-beam irradiation test. (N=3) Testing conditions Appearance pH

PuraStat@ control Clear and viscous 2.2

PuraStat@ irradiated (X-ray) at 25 kGy Clear and viscous 2.3

PuraStat@ irradiated (X-ray) at 40 kGy Clear and viscous 2.3

PuraStat@ irradiated (e-beam) at 25 kGy Clear and viscous 2.2

PuraStat@ irradiated (e-beam) at 40 kGy Clear and viscous 2.2

The appearance and pH of PuraStat@ after X-ray or e-beam irradiation were only

slightly changed or remained unchanged. The pH of PuraStat@ control was 2.2. The pH

of PuraStat@ after X-ray irradiation at 25 kGy and at 40 kGy were 2.3. The pH of

PuraStat@ after e-beam irradiation at 25 kGy and at 40 kGy were 2.2.

The appearance and pH of IEIK13 1.3% after X-ray or e-beam irradiation are

also shown in Table 4.

Table 4. Appearance and pH ofIEIK13 1.3% after X-ray or e-beam irradiation (N=3). Testing conditions Appearance pH

1EIK13 control Clear and viscous 3.0

1EIK13 irradiated (X-ray) at 25 kGy Clear and viscous 3.0

1EIK13 irradiated (X-ray) at 40 kGy Clear and viscous 3.0

1EIK13 irradiated (e-beam) at 25 kGy Clear and viscous 3.0

1EIK13 irradiated (e-beam) at 40 kGy Clear and viscous 3.0

The appearance and pH of1EIK13 1.3% after X-ray or e-beam irradiation were

not changed. The pH of1EIK13 1.3% control was 3.0. The pH of1EIK13 1.3% after X

ray or e-beam irradiation at 28 kGy and 40 kGy were 3.0.

The appearance and pH of KLD12 1.3% after X-ray or e-beam irradiation are

also shown in Table 5.

Table 5. Appearance and pH of KLD12 1.3% after X-ray or e-beam irradiation (N=3). Testing conditions Appearance pH

KLD12 control Clear and viscous 2.2

KLD12 irradiated (X-ray) at 25 kGy Clear and viscous 2.2

KLD12 irradiated (X-ray) at 40 kGy Clear and viscous 2.2

KLD12 irradiated (e-beam) at 25 kGy Clear and viscous 2.2

KLD12 irradiated (e-beam) at 40 kGy Clear and viscous 2.2

The appearance and pH of KLD12 1.3% after X-ray or e-beam irradiation were

not changed. The pH of KLD12 1.3% control was 2.2. The pH of KLD12 1.3% after X

ray or e-beam irradiation at 25 kGy and 40 kGy were 2.2.

The appearance and pH of QLEL12 0.15% after X-ray or e-beam irradiation are

also shown in Table 6.

Table 6. Appearance and pH of QLEL12 0.15% after X-ray and e-beam irradiation (N=3). Testing conditions Appearance pH

QLEL12 control Clear and viscous 7.0

QLEL12 irradiated (Gamma) at 25 kGy Clear and watery 6.5

QLEL12 irradiated (Gamma) at 40 kGy Clear and watery 5.9

QLEL12 irradiated (X-ray) at 25 kGy Clear and watery 6.3

QLEL12 irradiated (X-ray) at 40 kGy Clear and watery 5.9

QLEL12 irradiated (e-beam) at 25 kGy Clear and watery 6.6

QLEL12 irradiated (e-beam) at 40 kGy Clear and watery 6.5

The appearance of QLEL12 after gamma, X-ray or e-beam irradiation was

significantly altered. The pH of the QLEL12 control was 7.0. The pH's of QLEL12 after

gamma irradiation at 25 and 40 kGy were 6.5 and 5.9, respectively, those after X-ray

irradiation at 25 and 40 kGy were 6.3 and 6.5, respectively, and those after e-beam

irradiation at 25 and 40 kGy were 6.6 and 6.5, respectively. QLEL12 solutions became

watery after gamma, X-ray or e-beam irradiation.

In summary, RADA16, IEIK13 and KLD12 remained unchanged after gamma, X

ray and e-beam irradiation at around 25-40 kGy, but QLEL12 was not. Therefore, unlike

QLEL12, RADA16, IEIK13, and KLD12 can be sterilized using a gamma, X-ray and e

beam irradiation techniques.

Example6: Characterization by HPLC and Mass Spectrometry

The HPLC tests for RADA16, IEIK13, and QLEL12 were performed before and

after gamma, X-ray and e-beam irradiation, and the results for their major peptide

content were listed in Tables 7-10.

Table 7. HPLC test for major peptide content of PuraStat©(RADA16 2.5%) before and after gamma-irradiation (N=3, mean SD).

Testing conditions Major peptide content (%) PuraStat@ control 78.3 1.7

PuraStat@ irradiated (gamma) at 23 kGy 75.5 1.4*

PuraStat@ irradiated (gamma) at 25 kGy 74.6 0.7*

PuraStat@ irradiated (gamma) at 28 kGy 68.5 0.5*

PuraStat@ irradiated (gamma) at 40 kGy 69.7 0.5*

*: denotes if significantly lower than the data of PuraStat@ control (if p < 0.05, two tailed Student's t-test).

Table 8. HPLC test for major peptide contents of PuraStat@ (RADA16 2.5%) before and after X-ray and e-beam irradiation (N=3, mean SD).

Testing conditions Major peptide content (%)

PuraStat© control 78.3 1.7

PuraStat irradiated (X-ray) at 25 kGy 73.8 1.7*

PuraStat irradiated (X-ray) at 40 kGy 71.9 0.8*

PuraStat irradiated (e-beam) at 25 kGy 76.0 0.9*

PuraStat@irradiated (e-beam) at 40 kGy 70.0 2.4*

*: denotes if significantly lower than the data of PuraStat@ control (if p < 0.05, two tailed Student's t-test).

Table 9. HPLC test for major peptide contents of IEIK13 1.3% before and after gamma, X-ray, and e-beam irradiation (N=3, mean SD). Testing conditions Major peptide content (%)

1EIK13 control 99.6 ±0.0

IEIK13 irradiated (gamma) at 40 kGy 99.8 ±0.2

1EIK13 irradiated (X-ray) at 25 kGy 99.8 ±0.1

1EIK13 irradiated (X-ray) at 40 kGy 99.9 ±0.1

1EIK13 irradiated (e-beam) at 25 kGy 99.6 ±0.2

1EIK13 irradiated (e-beam) at 40 kGy 99.8 ±0.1

Table 10. HPLC test for major peptide contents of QLEL12 0.15% before and after gamma, X-ray, and e-beam irradiation (N=3, mean SD).

Testing conditions Major peptide content (%)

QLEL12 control 90.5 1.3

QLEL12 irradiated (gamma) at 40 kGy 40.7 3.1*

QLEL12 irradiated (X-ray) at 25 kGy 57.0 4.9*

QLEL12 irradiated (X-ray) at 40 kGy 47.3 1.3*

QLEL12 irradiated (e-beam) at 25 kGy 51.9 7.3*

QLEL12 irradiated (e-beam) at 40 kGy 55.6 3.0*

*: denotes if significantly lower than the data of QLEL12 control (if p < 0.05, two tailed Student's t-test).

The major peptide content of RADA16 control was 78.3%. The major peptide

contents of RADA16 after gamma irradiation at 23, 25, 28 and 40 kGy were 75.5, 74.6,

68.5 and 69.7%, respectively. The major peptide contents of RADA16 after X-ray

irradiation at 25 and 40 kGy were 73.8 and 71.9%, respectively. The major peptide

contents of RADA16 after e-beam irradiation at 25 and 40 kGy were 76.0 and 70.0%,

respectively.

The major peptide content of IEIK13 control was 99.6%. The major peptide

content of IEIK13 after gamma irradiation at 40 kGy was 99.8%. The major peptide

contents of IEIK13 after X-ray irradiation at 25 and 40 kGy were 99.8 and 99.9%,

respectively. The major peptide contents of IEIK13 after e-beam irradiation at 25 and 40

kGy were 99.6 and 99.8%, respectively.

The major peptide content of QLEL12 control was 90.5%. However, QLEL12

showed significant decrease in its major peptide content after irradiation sterilization.

The major peptide content of QLEL12 after gamma irradiation at 40 kGy was 40.7%.

The major peptide contents of QLEL12 after X-ray irradiation at 25 and 40 kGy were

57.0 and 47.3%, respectively. The major peptide contents of QLEL12 after e-beam

irradiation at 25 and 40 kGy were 51.9 and 55.6%, respectively. These results

demonstrate that RADA16 and IEIK13 remain relatively unchanged after gamma, X-ray

and e-beam irradiation compared to QLEL12.

The measured molecular weight of RADA16 was 1712, which matches its

calculated molecular weight (Figure 2). The mass spec analysis demonstrated that

RADA16 was not degraded after gamma, X-ray and e-beam irradiation (Figures 2-4).

However, RADA16 completely degraded during autoclave treatment.

Irradiation is a cold temperature sterilization technique unlike autoclaving.

Although both sterilization techniques provide high energy (i.e., radiation and heat) to

self-assembling peptides such as RADA16 during sterilization, irradiation sterilization

did not induce substantial degradation of RADA16 molecules.

However, all gamma, X-ray, and e-beam irradiated PuraStat@ mass spectra

exhibited some peaks that were not observed before irradiation, while no notable

difference was observed among them. The summary of the mass spectra is listed in

Table 11.

Table 11. Mass-spectrometry data of PuraStat@ after irradiation sterilization process.

Mz Mw at Mw at Mw at Estimated component Control Irradiated

n=1 n=2 n=3 PuraStat@* PuraStat@

502 501 ARADA-NH 2 (SEQ ID NO:5) Yes Yes

572 1712 Ac-(RADA) 4-NH 2 Yes Yes

(SEQ ID NO:1)

665 1328 ARADARADARADA-NH 2 Yes Yes

(SEQ ID NO:6)

836 1670 (RADA) 4-NH 2 No Yes

(SEQ ID NO:7)

857 1712 Ac-(RADA) 4-NH 2 Yes Yes

(SEQ ID NO:1)

916 915 ARADARADA-NH 2 Yes Yes

(SEQ ID NO:8)

1100 1100 ADARADARADA-NH 2 No Yes

(SEQ ID NO:9)

1143 1142 ARADARADARA Yes Yes

(SEQ ID NO:10)

1229 1228 Ac-RADARADARAD Yes Yes

(SEQ ID NO:11)

1329 1328 ARADARADARADA-NH 2 Yes Yes

(SEQ ID NO:6)

1513 1513 ADARADARADARADA-NH 2 No Yes

(SEQ ID NO:12)

1643 1642 Ac-RADARADARADARAD Yes Yes

(SEQ ID NO:13)

1670 1670 (RADA) 4-NH 2 No Yes

(SEQ ID NO:7)

1713 1712 Ac-(RADA) 4-NH 2 Yes Yes

(SEQ ID NO:1)

*:stored at 2-8 °C for about 4 years. #: all PuraStat samples were irradiated at 40 kGy (with gamma rays, X-rays, and e beam)

The control PuraStat@ showed Mz peaks at 572, 857, and 1713, which are

assigned to the major peptide, Ac-(RADA)4-NH2 (SEQ ID NO:1). Control PuraStat@ also

exhibited other Mz peaks at 502, 665/1329, 916, 1143, and 1229, which are estimated

as ARADA-NH2 (SEQ ID NO:5), ARADARADARADA-NH2 (SEQ ID NO:6),

ARADARADA-NH2 (SEQ ID NO:8), ARADARADARA (SEQ ID NO:10), Ac

RADARADARAD (SEQ ID NO:11), respectively.

Table 12 shows the pattern of degradation of Ac-(RADA)4-NH2 (SEQ ID NO:1)

when PuraStat@ is stored at 2-8 °C for about 4 years. From the pattern, we found that

degradation mainly occurs at the points between -RAD and A-.

On the other hand, the irradiated PuraStat@ showed additional Mz peaks at

836/1670, 1100, and 1513, which are estimated as (RADA)4-NH2 (SEQ ID NO:7),

ADARADARADA-NH2 (SEQ ID NO:9), and ADARADARADARADA-NH2 (SEQ ID

NO:12), respectively.

Table 13 shows the additional pattern of degradation of Ac-(RADA)4-NH2 (SEQ

ID NO:1) when PuraStat@ is sterilized with irradiation. Especially, the peaks at 836 and

1670 are ones of major additional peaks, which represent RADARADARADARADA-NH2

(SEQ ID NO:7). This means that irradiation can cause additional degradation of

PuraStat@ at the point between acetyl group (Ac) and RAD-. The peaks at 1100 and

1513 are also ones of major additional peaks, which represent ADARADARADA-NH2

(SEQ ID NO:9), and ADARADARADARADA-NH2 (SEQ ID NO:12). This means that

irradiation can cause additional degradation of PuraStat@ at the point between -R and

AD-.

Table 12. Pattern of degradation of Ac-(RADA)4-NH2 (SEQ ID NO:1) (PuraStat@ was stored at 2-8 °C for about 4 years)

Ac-RADARADARADARADA-NH2 (SEQ ID NO:1) (before degradation)

Ac-RAD / ARADARADARADA-NH2(SEQIDNO:6)

Ac-RADARAD(SEQIDNO:14) / ARADARADA-NH2(SEQIDNO:8)

Ac-RADARADARAD(SEQIDNO:11) / ARADA-NH2(SEQIDNO:5)

Ac-RAD / ARADARADARA(SEQIDNO:10) / DA-NH2

Ac-RADARADARADARAD (SEQ ID NO:13) / A-NH2

Table 13. Additional pattern of degradation of Ac-(RADA)4-NH2 when PuraStat@ is sterilized with irradiation

Ac-RADARADARADARADA-NH2 (SEQ ID NO:1) (before degradation)

Ac I RADARADARADARADA-NH2(SEIDNO:7)

Ac-R I ADARADARADARADA-NH2(SEQIDNO:12)

Ac-RADAR(SEQIDNO:14) / ADARADARADA-NH2(SEQIDNO:9)

Also, the molecular weight of IEIK13 was measured at 1622, which matches its

calculated molecular weight (Figure 5). The mass spec analysis demonstrated that

IEIK13 was only insubstantially degraded after gamma, X-ray and e-beam irradiation.

Furthermore, the molar mass of QLEL12 was measured at 1506, which matches

its calculated molar mass (Figure 6). However, the mass spec analysis demonstrated

that QLEL12 was significantly degraded after gamma, X-ray and e-beam irradiation.

Example 7: Rheological properties

Based on ISO 11137 (Sterilization of health care products - Radiation), radiation

sterilization methods can be used with 25 kGy or 15 kGy irradiation as the sterilization

dose to achieve a sterility assurance level, SAL, of 10-6.

The rheology results are shown in Figures 7 and 8 for PuraStat@ with gamma

irradiation sterilization before and after gelation, respectively. The determined

rheological results are listed in Tables 14-15.

Table 14. Results from frequency tests of PuraStat@ (RADA16 2.5%) with gamma irradiation at 0.1% of strain with 40 mm cone-plate

Storage Modulus G'at 1 Hz (Pa)

Sample # Gamma- Gamma- Gamma PuraStat@ irradiated irradiated irradiated control PuraStat@ at PuraStat@ at PuraStat@ at 23kGy 25kGy 40kGy

1 343.7 514.1 546.0 551.0

2 323.3 449.7 490.2 607.7

3 301.7 449.5 467.0 618.4

Mean 322.9 471.1* 501.0* 592.4*,$

SD 21.0 37.2 40.6 36.2

*:denotes if p <0.05 compared to control (two tailed Student's t-test). $: denotes if p <0.05 compared to the others (two tailed Student's t-test).

PuraStat@ gamma-irradiated at 23 kGy, 25 kGy, and 40 kGy showed higher

storage modulus than PuraStat© control (Figure 7 and Table 14). PuraStat@ gamma irradiated at 40 kGy showed significantly higher storage modulus than PuraStat@ gamma-irradiated at 23 and 25 kGy. Also, PuraStat@ gamma-irradiated at 25 kGy showed slightly higher storage modulus than PuraStat@ gamma-irradiated at 23 kGy.

This indicates that gamma-irradiation positively affected the rheological properties of

PuraStat@. PuraStat@ gamma-irradiated at 23 kGy (471.1 37.2 Pa), 25 kGy (501.0

40.6 Pa) and 40 kGy (592.4 36.2 Pa) exhibited 46%, 55%, and 83% increases in their

storage moduli, respectively, compared to PuraStat@ control (322.9 ±21.0 Pa).

Table 15. Results from frequency tests of PuraStat@ (RADA16 2.5%) with gamma irradiation after gelation. Samples were treated with DMEM for 20 min at 0.1% of strain with a 40 mm cone-plate

Storage Modulus G'at 1 Hz (Pa)

Sample# Gamma- Gamma- Gamma PuraStat@ irradiated irradiated irradiated control PuraStat@ at PuraStat@ at PuraStat@ at 40 23kGy 25kGy kGy

1 4454 8866 6885 14568

2 5280 8845 10395 10286

3 5183 8622 10148 12134

Mean 4972 8711* 9143* 12330*

SD 451 135 1959 2148

*:denotes if p < 0.05 compared to control (two tailed Student's t-test). $: denotes if p <0.05 compared to the others (two tailed Student's t-test).

Furthermore, after gelation triggered by simulated body fluid (i.e., DMEM buffer)

for 20 min, PuraStat@ gamma-irradiated at 23 kGy (8711 ±135 Pa), 25 kGy (9143

1959 Pa) and 40 kGy (12330 ±2148 Pa) exhibited 75%, 84%, and 148% increases in

their storage moduli, respectively, compared to PuraStat@ control (4972 ±451 Pa)

(Figure 8 and Table 11).

The rheology results are shown in Tables 16 and 17 for PuraStat@ with X-ray

irradiation sterilization before and after gelation, respectively.

Table 16. Results from frequency tests of PuraStat@ (RADA16 2.5%) with X-ray irradiation at 0.1% of strain with 40 mm cone-plate

Storage Modulus G' at 1 Hz (Pa) Sample #

PuraStat@ control X-ray-irradiated X-ray-irradiated PuraStat@ at 25 kGy PuraStat@ at 40 kGy

1 343.7 448.3 610.2

2 323.3 429.3 632.5

3 301.7 471.7 578.9

Mean 322.9 449.8* 607.2*,$

SD 21.0 21.2 27.0

*:denotes if p < 0.05 compared to control (two tailed Student's t-test). $: denoted if p <0.05 compared to the others (two tailed Student's t-test).

PuraStat*X-ray-irradiated at 25 kGy and 40 kGy also showed higher storage

modulus than PuraStat© control. PuraStat*X-ray-irradiated at 40 kGy showed

significantly higher storage modulus that PuraStat*X-ray-irradiated at 25 kGy. This

indicates X-ray irradiation positively affected the rheological properties of PuraStat©.

PuraStat irradiated at 25 kGy (449.8 21.2) and 40 kGy (607.2 ±27.0 Pa) exhibited 39%

and 88% increases in their storage moduli, respectively, compared to PuraStat© control

(322.9 ±21.0 Pa) (Table 17).

Table 17. Results from frequency tests of PuraStat© (RADA16 2.5%) with X-ray irradiation after gelation. Samples were treated with DMEM for 20 min. at 0.1% of strain with a 40 mm cone-plate.

Storage Modulus G'at 1 Hz (Pa) Sample #

PuraStat© control X-ray-irradiated X-ray-irradiated PuraStat© at 25 kGy PuraStat at 40 kGy

1 4454 9563 12832

2 5280 6404 10611

3 5183 9522 10294

Average 4972 8497* 11246*

SD 451 1812 1383

*:denotes if p < 0.05 compared to control (two tailed Student's t-test).

Furthermore, after gelation triggered by simulated body fluid (i.e., DMEM buffer)

for 20 min, PuraStat*X-ray-irradiated at 25 kGy (8497 ±1812 Pa) and 40 kGy (11246

1383 Pa) exhibited 71% and 126% increases in their storage moduli, respectively,

compared to PuraStat© control (4972 ±451 Pa) (Table 17).

The rheology results are shown in Tables 18 and 19 for PuraStatlwith e-beam

irradiation sterilization before and after gelation, respectively.

Table 18. Results from frequency tests of PuraStat©(RADA16 2.5%) with e-beam irradiation at 0.1% of strain with 40 mm cone-plate.

Storage Modulus G'at 1 Hz (Pa) Sample #

PuraStat©control e-beam-irradiated e-beam-irradiated PuraStat© at 25 kGy PuraStat at 40 kGy

1 343.7 348.1 349.6

2 323.3 356.1 371.5

3 301.7 324.0 338.4

Mean 322.9 342.7 353.2

SD 21.0 16.7 16.8

PuraStat e-beam-irradiated at 25 kGy and 40 kGy, however, did not show a

significant change in storage modulus compared to PuraStat control. This indicates e

beam irradiation sterilization does not have a major effect on the rheological properties

of PuraStat©. However, although the p values did not show statistical significance (i.e., p

> 0.05), PuraStat e-beam irradiated at 25 kGy (342.7 ±16.7 Pa) and 40 kGy (353.2±

16.8 Pa) exhibited 6% and 9% increases in their storage moduli, respectively, compared

to PuraStat© control (322.9 ±21.0 Pa) (Table 18).

Table 19. Results from frequency tests of PuraStat©(RADA16 2.5%) with e-beam irradiation after gelation. Samples were treated with DMEM for 20 min at 0.1% of strain with 40 mm cone-plate.

Storage Modulus G'at 1 Hz (Pa) Sample #

PuraStat©control e-beam-irradiated e-beam-irradiated PuraStat© at 25 kGy PuraStat'at 40 kGy

1 4454 3623 4918

2 5280 4830 9327

3 5183 3542 7875

Average 4972 4007 7373

SD 451 715 2247

After gelation triggered by simulated body fluid (i.e., DMEM buffer) for 20 min,

PuraStat e-beam-irradiated at 25 kGy and 40 kGy did not show significant difference

(i.e., the p values were higher than 0.05) in their storage moduli compared to PuraStat©

control (Table 19).

PuraStat© demonstrated shear thinning property at high shear rate and

thixotropic behavior suggesting slow rheological property recovery when high shearing

stopped (Figure 9). From these properties of PuraStat@ , the stiffness of PuraStat© can

be lowered for easier handling during application to patients and stiffness can then

slowly recover to initial values after application. These intrinsic thixotropic properties of

PuraStatlwere not changed, even after gamma irradiation at 23 and 25 kGy, while

irradiated PuraStat© showed higher storage modulus than PuraStat© control.

Because the peptides' molecular structure was not substantially changed

considering the results of HPLC and Mass Spectrometry, the assembled nanofibrous

structure of the peptides could be a factor to increase their rheological properties. The

rheological properties of self-assembling peptide solution might increase when self

assembled peptide structure is more organized. By way of a non-binding theory, high

energy from irradiation can make peptide molecules move slightly to have more

organized nanofibrous structure resulting in improved rheological properties.

The increased rheological properties were at least partially reversed by repeated

high shearing and returned back close to the original levels. After thoroughly shearing

them twice at 1000 s-1 for 1 minute, PuraStat© samples irradiated at 23 kGy and 25 kGy

showed gradual decrease in their storage modulus from 484.5 Pa to 410.9 Pa and

514.3 Pa to 426.5 Pa, respectively, while PuraStat@control did not show significant

change in its storage modulus (Figure 9). It could be expected that the rheological

properties of irradiated PuraStat© become closer to those of PuraStat© control with more

shearing. Therefore, gamma irradiation enhances the structure of self-assembled

nanofibers to increase their rheological properties without a detectable change in

peptide molecular structure degradation or crosslinking.

Example 8: Bioburden Testing

A. Collection of Samples. Upon irradiation, 50 samples were collected as follows:

10 samples for bioburden tests inside the packages (collecting 4 at the beginning, each

3 at the middle and the end of the packaging operation); 10 samples for bioburden tests

of the filling liquid in the syringe (collecting 4 at the beginning, each 3 at the middle and

the end of the packaging operation); spare samples, e.g., 30 samples, may be collected

in case extra testing may be required.

B. Viable Bacteria Count Test for Sterilization Validation. The sample was

aseptically prepared in a clean bench, and all instruments and solvents to be used were

sterilized.

For blister-packaged products, the following procedure was repeated twice with

one sealed package to make a total 100 mL of sample solution. A syringe was used to

inject 50 mL of rinsing fluid for the sterilization test (USP Fluid D) into the inside of the

package. A sample were shaken thoroughly and allowed to stand to reduce bubbles.

Then, the outside opening of the package was sterilized by lightly passing the opening

through a flame without heating the contents. Then, a syringe or another suitable

method was used to extract all the injected solution from the package, which was then

collected in a heat-resistant bottle. With a sealed blister package containing about 100

CPU of spores purified from a standard strain such as Bacillus subtilis (NBRC3134), a

recovery rate and a correction coefficient based on the recovery rate were calculated in

advance from the viable bacteria count and the added bacteria count from a sample

which was prepared in the same manner as above. A correction coefficient was

calculated as 1/recovery rate (%) x 100. This correction coefficient was recalculated

when the sample preparation procedure was changed.

For testing content fluid, 1 mL of the content solution was mixed with 9 mL of

Soybean-Casein Digest Medium agar medium (SCD agar medium), and the gel finely dispersed to make 10 mL of sample solution.

For blister package products, 100 mL of the sample solution per culture medium

was used and the test was conducted using the membrane filter method of the

Japanese Pharmacopoeia Microbial Limit Test.

For testing content fluid, 1 mL of the sample solution was added per culture

medium and conduct the test using the agar plate dilution method of the Japanese

Pharmacopoeia Microbial Limit Test. This test was repeated 10 times to obtain 10

culture media plates corresponding to 10 mL of sample solution.

The cultures were maintained at 30°C-35 0C for 3-5 days (or longer) on SCD

agar medium. As a general rule, the cultures were observed at least once every

operating day during the culture period and on the final measurement day.

After completion of the culture, the actual measured values of the colonies of

SCD agar medium were converted with the following calculation:

(1) For blister package products Viable bacteria count in a blister package = Viable bacteria count in 100 mL of sample liquid x Correction coefficient (2) For Content fluid Viable bacteria count in 1 mL of content liquid = Total viable bacteria count in 10 mL equivalent to sample solution (10 culture media plates)

Remarks: Among the test method for the content fluid, the value of the SIP (aliquot)

used for calculation were determined to be 1/5 = 0.2, not the total amount when testing

with a 5-mL product by the test method equivalent to 1 mL of content liquid. * SIP

(Sample Item Portion) is equivalent to an aliquot (defined portion of the healthcare product used for testing).

Example 9: Experimental design of PuraStat@ sterilization by Gamma irradiation

Irradiation conditions--PuraStat© samples (Lot# 17C09A30) were irradiated with

gamma rays at 25, 28 and 40 kGy with Gammacell 220© High Dose Rate Co-60

Irradiator (MDS Nordion, Ottawa, Canada). The run dose rate and duration time for 40

kGy irradiation were 6.30 kGy/hr and 6 hours 20 minutes 58 seconds, respectively. The

run dose rate and duration time for 28 kGy irradiation were 4.40 kGy/hr and 6 hours 22

minutes 31 seconds, respectively. The run dose rate and duration time for 25 kGy

irradiation were 6.58 kGy/hr and 3 hours 47 minutes 42 seconds, respectively.

Methods and test results: The appearance of PuraStat~was observed after each

test. The pH of PuraStat© was tested using an Accumet AB15 pH meter (Fisher

Scientific).

HPLC tests were performed to evaluate the major peptide content after irradiation tests.

Agilent HPLC 1100 (Agilent Technologies) was used for this study. Column temperature

was kept at 25 °C.

Solvent A was water with 0.1% TFA and solvent B was 80% Acetonitrile with 0.1%

TFA. Gradient of solvent B was controlled from 10% to 40% in 20 min and 40% for

another 5 min at 25 °C. Agilent Zorbax 300SB-C18 column (4.6 mm X 250 mm, 5 tm,

300 A) was used for this test. PuraStat@ (RADA16 2.5%) (40 mg) were mixed with 10

pL of DH2O and 500 pL of formic acid and vortexed. And the mixture was mixed with

DH2O (4,450 pL) and vortexed. 20 tL of samples were injected using an Agilent

autosampler.

Mass spectrometry tests were carried out to investigate the degradation of the

peptides after irradiation sterilization. Agilent LC/MSD ion trap mass spectrometer was

used for this study. The sample solutions were prepared as described above for the

HPLC samples. Each sample was injected at 9 pL/min with a syringe pump. Mass

spectrum was recorded for 1 minute. The molecular weight of PuraStat@ (Ac-(RADA)4

NH2) (SEQ ID NO:1) is 1712, which matches its calculated molecular weight from the

major three peaks at m/3=572, m/2=857, and m=1713 in all the spectra of control and

irradiated PuraStat@ samples.

The assigned peaks to Ac-RADARADARADARADA-NH2 (SEQ ID NO:1) are,

M/z= 572 = (Mw +3)/3, so the calculated Mw = 1713

M/z= 857 = (Mw +2)/2, so the calculated Mw = 1712

M/z= 1713 = Mw +1, so the calculated Mw = 1712

The rheological properties of PuraStat ©samples before and after gamma

sterilization were evaluated using a rheometer (Discovery HR 1, TA Instruments) at

37°C. Flow tests were carried out with 20 mm plate-plate geometry and 800 pm of gap

distance at a shear rate range of 0.001 1/sec to 3,000 1/sec at 37°C. Sample solution

(350 pL) was placed on the rheometer plate and excess solution was gently removed;

measurements were performed after 2 minutes of relaxation time at 37°C. The viscosity

(h) were recorded from very low shear rate (0.001 1/sec) to high shear rate (3000 1/sec).

L929 neutral red uptake tests were performed to investigate the cytotoxicity of

the irradiated PuraStat@. These tests were performed by Toxikon located in Bedford

MA, USA. The study was done based upon ISO 10993-5, 2009, Biological Evaluation of

Medical Devices - Part 5: Tests for in Vitro Cytotoxicity and ISO 10993-12, 2012,

Biological Evaluation of Medical Devices - Part 12: Sample Preparation and Reference

Materials. The biological reactivity of a mammalian cell monolayer, L929 mouse

fibroblast, in response to the test article extract was determined. The test article extract

was obtained with serum-supplemented Minimum Essential Medium (MEM) at the ratio

0.2 g of article per mL. Extraction was done for 24± 2 hours at 37± 1 C. Positive

control (Natural Rubber) and negative control (Negative Control Plastic) articles and an

untreated control (blank) were prepared to verify the proper functioning of the test

system. The test article and control article extracts were used to replace the

maintenance medium of the cell culture. The test article extract was tested at the 100%

(neat) concentration. All cultures were incubated in at least 6 replicates for 24 to 26

hours. At 37± 1 C, in a humidified atmosphere containing 5 ±1 % carbon dioxide

(C02). The viability of cells following the exposure to the extracts was measured via

their capacity to uptake a vital dye, Neutral Red. This dye was added to the cells to be

actively incorporated in viable cells. The number of viable cells correlates to the color

intensity determined by photometric measurements at 540 nm after extraction.

The viability of cells exposed to the negative control article and positive control

article extracts needs to be greater than or equal to 70% and less than 70% of the

untreated control, respectively, to confirm the validity of the assay. The test article meets the requirements of the test if the viability % is greater than or equal to 70% of the untreated control.

We tested only PuraStat@ samples irradiated at the highest dose (i.e., 40 kGy)

considering they represent all the irradiated samples, because they should have more

effects, if yes, on cytotoxicity than those irradiated at lower doses.

Table 20. Summary Table of Irradiation Sterilization Tests

conditions Appearance pH andMS Rheology Cytotoxicity

PuraStat@ Clear and 2.2 Control Control Not toxic (Control) viscous PuraStat© with Clear and 2.3 ~5% Gamma at 25kGy viscous degrade

PuraStat© with Clear and ~15%. Gamma at 28k or viscous 2.3 degrade equivalent Not toxic 40 kGy

The amount of peptide prior to the irradiation. After up to 25 kGy of irradiation,

PuraStat's degradation did not exceed 5% of the amount of peptide prior to the

irradiation.

From the pattern of degradation of control PuraStat©, we found that degradation

mainly occurs at the points between -RAD and A-. From the additional pattern of

degradation of Ac-(RADA)4-NH2 (SEQ ID NO:1) when PuraStat© is sterilized with

irradiation, we found that irradiation can cause additional degradation at the point

between -R and AD-.

Overall, the rheological property of PuraStat© with gamma irradiation was equivalent

to PuraStat© control. All the irradiated PuraStat© and PuraStat© control samples meet

the requirements of the test and are not considered to have a cytotoxic potential.

HPLC and LCMS results in each peak

PuraStat@ current product (non-irradiated) PuraStat@ proposed product (irradiated)

HPLC LC-MS HPLC LC-MS

Representative Representative Peak tR Area Area % Peak tR Area Area

% m/z m/z

1 6.37 25.70 4.39 1329(666) 1 6.35 8.20 1.90 1329(666)

2 6.73 8.49 1.44 1229(615) 2 6.73 2.90 0.67 1229(615)

3 7.15 9.98 1.71 1300(651) 3 7.27 12.00 2.78 1143(572)

1143(572) 4 7.30 9.29 1.59 4 7.56 2.29 0.53 1671(836) 893

7.75 4.75 0.81 1671(836), 5 7.71 2.34 0.54 1671(836) 1713(857)

6 8.01 7.49 1.28 1713(857), 750 6 7.90 7.62 1.77 16713(857)

1643(823), 1643(823), 7 8.18 6.95 1.19 1713(857) 7 8.16 4.06 0.94 1713(557), 1513

1643(823), 1643(823), 8 8.29 2.15 0.36 1713(857) 8 8.27 7.95 1.84 1713(557), 1513

9 8.45 473.23 80.80 1713(857) 9 8.47 348.78 80.89 1713(857)

10 8.80 11.89 2.03 1557,783,857 10 8.77 12.95 3.00 1557,783

11 8.93 4.54 0.77 783,857 11

12 9.06 4.85 0.82 839, 1713(857) 12 9.00 10.46 2.43 839, 1713(857)

13 10.00 16.26 2.77 482,839,891 13 9.96 11.61 2.69 839,482

Table 21. The HPLC and LC-MS analysis for two Product Forms

Table 21 below shows results of HPLC tests performed to evaluate the major peptide

content before (control) and after Gamma irradiation.

Agilent HPLC 1100 (Agilent Technologies) was used for this study. Column

temperature was kept at 25 °C. Agilent Zorbax 300SB-C18 column (4.6 mm X 250 mm,

5mm, 300 A) was used for this test. Solvent A was water with 0.1% TFA and solvent B

was 80% Acetonitrile with 0.1% TFA. Gradient of solvent B was controlled from 10% to

40% in 20 min and 40% for another 5 min at 25 °C. PuraStat@ (40 mg) were mixed with

10 pL of DH2O and 500 pL of formic acid and vortexed. And the mixture was mixed with

DH2O (4,450 pL) and vortexed. 20 mL of samples were injected using an Agilent

autosampler. Results are shown in following Table 23.

Table 23.

PuraStat@ control PuraStat@ irradiated above 25 kGy Majo Pept de 83,8+/-1% 81,3 +/- 0,7% Manufacturing 75% and more Specification

Findings

Overall, the major peptide content of PuraStat@ decreased with gamma irradiation

methods and the extent of decrease was more significant with higher dose. After up to

40 kGy of irradiation, PuraStat@'s degradation did not exceed 15% of the amount of

peptide prior to the irradiation. After up to 25 kGy of irradiation, PuraStat@'s degradation

did not exceed 5% of the amount of peptide prior to the irradiation.

From the pattern of degradation of control PuraStat@, we found that degradation

mainly occurs at the points between -RAD and A-. From the additional pattern of

degradation of Ac-(RADA)4-NH2 when PuraStat@ is sterilized with irradiation, we found

that irradiation can cause additional degradation at the point between -R and AD-.

Overall, the rheological property of PuraStat@ with gamma irradiation was

equivalent to PuraStat@ control. All the irradiated PuraStat@ and PuraStat@ control

samples met the requirements of the test and were not considered to have a cytotoxic

potential.

SEQUENCE LISTING

<110> 3-D MATRIX, LTD.

<120> STERILIZATION OF SELF-ASSEMBLING PEPTIDES BY IRRADIATION

<130> 3DMUS-2021-02-IRR-PCT

<140> PCT/US21/24954 <141> 2021-03-30

<150> 63/002,882 <151> 2020-03-31

<160> 15

<170> PatentIn version 3.5

<210> 1 <211> 16 <212> PRT <213> Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic peptide

<220> <223> N-term Acetylated

<220> <223> C-term Amidated

<400> 1 Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala 1 5 10 15

<210> 2 <211> 12 <212> PRT <213> Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic peptide

<220> <223> Synthetically produced

<220> <223> N-term Acetylated

<220> <223> C-term Amidated

<400> 2 Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu Asp Leu 1 5 10

<210> 3 <211> 13 <212> PRT <213> Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic peptide

<220> <223> Synthetically produced

<220> <223> N-term Acetylated

<220> <223> C-term Amidated

<400> 3 Ile Glu Ile Lys Ile Glu Ile Lys Ile Glu Ile Lys Ile 1 5 10

<210> 4 <211> 12 <212> PRT <213> Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic peptide

<220> <223> Synthetically produced

<220> <223> N-term Acetylated

<220>

<223> C-term Amidated

<400> 4 Gln Leu Glu Leu Gln Leu Glu Leu Gln Leu Glu Leu 1 5 10

<210> 5 <211> 5 <212> PRT <213> Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic peptide

<220> <223> Synthetically produced

<220> <223> C-term Amidated

<400> 5 Ala Arg Ala Asp Ala 1 5

<210> 6 <211> 13 <212> PRT <213> Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic peptide

<220> <223> Synthetically produced

<220> <223> C-term Amidated

<400> 6 Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala 1 5 10

<210> 7 <211> 16 <212> PRT

<213> Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic peptide

<220> <223> Synthetically produced

<220> <223> C-term Amidated

<400> 7 Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala 1 5 10 15

<210> 8 <211> 9 <212> PRT <213> Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic peptide

<220> <223> Synthetically produced

<220> <223> C-term Amidated

<400> 8 Ala Arg Ala Asp Ala Arg Ala Asp Ala 1 5

<210> 9 <211> 11 <212> PRT <213> Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic peptide

<220> <223> Synthetically produced

<220>

<223> C-term Amidated

<400> 9 Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala 1 5 10

<210> 10 <211> 11 <212> PRT <213> Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic peptide

<220> <223> Synthetically produced

<400> 10 Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala 1 5 10

<210> 11 <211> 11 <212> PRT <213> Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic peptide

<220> <223> Synthetically produced

<220> <223> N-term Acetylated

<400> 11 Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp 1 5 10

<210> 12 <211> 15 <212> PRT <213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic peptide

<220> <223> Synthetically produced

<220> <223> C-term Amidated

<400> 12 Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala 1 5 10 15

<210> 13 <211> 15 <212> PRT <213> Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic peptide

<220> <223> Synthetically produced

<220> <223> N-term Acetylated

<400> 13 Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp 1 5 10 15

<210> 14 <211> 7 <212> PRT <213> Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic peptide

<220> <223> Synthetically produced

<220> <223> N-term Acetylated

<400> 14

Arg Ala Asp Ala Arg Ala Asp 1 5

<210> 15 <211> 5 <212> PRT <213> Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic peptide

<220> <223> Synthetically produced

<220> <223> N-term Acetylated

<400> 15 Arg Ala Asp Ala Arg 1 5

Claims (1)

1. A method of sterilizing a self-assembling peptide (SAP) solution, the method

comprising:

(a) placing one or more containers with the solution of self-assembling peptide

into an irradiation machine, said self-assembling peptide capable of

forming a hydrogel when applied to a biological tissue at about neutral pH;

and

(b) exposing the container to gamma ray, X-ray and/or e-beam irradiation at a

predetermined dose so that the self-assembling peptide solution is

sterilized to a pre-determined Sterility Assurance Level (SAL) without

substantial degradation of the peptide, wherein the concentration of the

degradation products of the full-length peptide ("major peptide") in the

solution post-irradiation ranges from 0.1% to 20% by weight while its

desired physical properties are maintained substantially at the same level

or improved;

wherein the peptide is selected from the group consisting of RADA16, KLD12,

and IEIK13; and

wherein the desired physical properties are storage modulus and/or viscosity.

2. The method of claim 1, wherein the dose is 15 - 50 kGy .

3. The method of claim 1 or claim 2, wherein the solution is irradiated by X-ray or

e-beam.

4. The method of claim 2 or claim 3, wherein the peptide solution contains about

2.5% w/v of RADA16, and the dose is 15-24 kGy.

5. The method of any one of claims 1-4, wherein the amount of total ("over-all")

peptides that are degraded after the irradiation does not exceed 5% by weight of the

amount of the self-assembling peptide prior to the irradiation.

6. The method of claim 1, wherein the pre-irradiation bioburden of the self

assembling peptide solution is 9 CFU per product unit or less.

7. The method of any one of claims 1-6, wherein irradiation dose achieves

sterility assurance level (SAL) of at least 10-6.

8. The method of any one of claims 1-7, wherein the pH of the solution pre- and

post-irradiation ranges from about 1.8 to 3.5.

9. The method of any one of claims 1-8, wherein the one or more containers

is/are plastic syringe(s).

10. The method of any one of claims 1-9, wherein the storage modulus of the

hydrogel is increased at least by 10% post-irradiation.

11. The method of claim 1, further comprising: c) shearing the solution to reduce

or restore its storage modulus.

12. A sterilized solution of self-assembling peptide made by the method of any

one of claims 1-11.

13. A method of using the sterilized solution of claim 12, comprising applying the

solution to a biological tissue.

14. The method of claim 13, wherein the sterilized solution is applied during

surgery or after trauma involving bleeding.

15. The method of claim 14, wherein the sterilized solution is applied to an

internal site in a human subject.

16. The method of claim 1, wherein the self-assembling peptide solution exhibits

a post-irradiation mass spectrometric (MS) profile having major Mz peaks at

836/1670, 1100, and 1513 m/z.

17. Use of a sterilized solution of the self-assembling peptide for treating or

preventing a disease or condition, wherein the sterilized solution is obtained by the

method of any one of claims 1-11.

Y-irradiation

Figure 1B

Figure 1A PuraStat@ m/z m/z

#(1-88) 0.0-1.0min +MS, #(1-82) 0.0-1.0min +MS, 1712.2

2000

2000

1800

1713.0

1750

1600

1328.2 1500

1400

1328.7

1250

1200

1143.2

1000 1000

915.1

856.9 857.3

800 750

665.3

665.2

572.5

572.1 600 502.5

502.4 500

414.4

414.3

400

250

Figure 2A Figure 2B

200 (B)

Intens Intens

x106 2.5 2.0 1.5 1.0 0.5 0.0 x10 5 5 4 3 2 1 0 m/z

#(1-456) 0.0-5.0min +MS, 2000

1800

1600

1400

1229.6

1200

1000

845.5

800

615.5

600

502.5

432.4

400

317.4

Figure 2C

200

Intens x107

3 2 1 0 m/z +MS, 0.0-1.3min #(1-117) m/z +MS, 0.0-1.0min #(1-91)

2000 2000

1750 1750 1712.1 1712.1

1500 1500

1328.1 1328.1

1250 1250

1143.0 1143.0

1000 1000

914.9 914.9

856.6 856.5

750 750

664.4 664.4

571.4 571.3

501.8

500 500

111.61 402.7 384.7 Hay

250 250

Figure 3A Figure 3B

(B)

Intens Intens x106 3.0 2.5 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 x10 m/z m/z +MS, 0.0-1.0min #(1-96) +MS, 0.0-1.0min #(1-91)

2000 2000

1750 1750 1712.1 1713.1

1500 1500

1328.0 1328.1

1250 1250

1142.9 1143.0

1000 1000

914.9 914.9

856.5 856.4

750 750

664.8 664.3

571.3 571.3

501.7

500 500

384.6 384.6

250 250

Figure 4A Figure 4B

Intens. Intens.

x106 x106 5 4 3 2 1 0 4 3 2 1 0

+MS, 0.0-1.0min #(1-90) m/z +MS, 0.0-1.0mir #(1-90) m/z

2000 2000

1750 1750

1622.4 1622.4

1509.4

1494.4

1500 1500

1381.3

1338.4

1252.3

1250 1225.3 1250

1139.2

1000 984.1 1000

811.8 811.6

755.1 755.1

750 750

627.0 614.0

541.4 541.4

500 500

319.1

250 250

Figure 5A Figure 5B

Intens Intens 1.0 0.8 0.6 0.4 0.2 0.0 x106 x105 6 4 2 0 m/z m/z +MS, 0.0-1.0min #(1-89) +MS, 0.0-1.0min #(1-88)

2000 2000

1750 1750

1622.4 1622.5

1509.4

1500 1500

1338.4 1338.4

1252.3

1250 1225.3 1250

1139.2

1097.3

984.1 1000 984.2 1000

811.8 811.8

755.2 755.1

750 750

669.6

613.0

541.5 541.4

500 500

250 250

Figure 5C Figure 5D

Intens. Intens.

4 3 2 0 3 2 0 55 1 1 x105 x10 m/z m/z +MS, 0.0-1.0min #(1-88) +MS, 0.0-1.0min #(1-88)

2000 2000

1750 1750

1622.3 1622.0

1509.3 1508.8

1500 1500

11381.3 1252. 1096.4 984.0 1251.8 1380.1

1250 1250

1000 1000

811.5 811.7

755.0 755.1

750 750

613.0 612.4

541.5

500 500

250 250

Figure 5E Figure 5F

Intens. Intens.

x105 4 3 2 1 0 5 x10 3 2 1 0

1750

1505.5 1500 m/z -MS, 0.0-1.0min #(3-81)

-MS, 0.0-1.1min #(1-97) 2195.8 m/z

2018.7

2000 2000

1846.6

1750 1750

1620.0

1563.1

1507.1 1507.1 1500 1500

1436.1

1250 1250

1086.0

1000 1000

752.9 753.1

750 750

675.9

618.0

560.1 560.1

502.1

500 500 444.1

326.1

268.1 268.0

250 250 210.0

Figure 6B Figure 6C

Intens. Intens 1.5 1.0 0.5 0.0 x104 x104 5 4 3 2 1 0 m/z 2191.5 m/z -MS, 0.0-1.1min #(1-96) 2194.3 -MS, 0.0-1.1min #(1-97)

2078.2

2000 2000

1842.4

1750 1750

1507.3 1505.8

1500 1500

1379.3

1321.4 1315.5

1261.4

1250 1250

1145.6

1087.6

1026.3

1000 1000 969.7

868.5

793.9 791.7

750 750

560.1 558.0

500 500

326.1

170.0 266.3 268.0

250 250 210.0

Figure 6D Figure 6E

Intens Intens.

2.5 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 x104 x10

-MS, 0.0-1.1min #(1-97)

2189.1 m/z

2016.7

2000

1840.6

1750

1500

1316.7

1250

1025.3

1000

867.0

791.1

750

557.9

500

266.3

250

169.9 Figure 6F

Intens 0.8 0.6 0.4 0.2 0.0 x104

10.0

PuraStat® Control PuraStat® 23KGy PuraStat® 25KGy PuraStat® 40KGy

Frequency (Hz)

1.0

0.1

1000 100 10

Figure 7

Figure 8 100000 10000 gelation after - control PuraStat A gelation after - 23KGy PuraStat 1000 gelation after - 25KGy PuraStat gelation after Las 40KGy PuraStat 100 1.0 10.0

0.1 Frequency (Hz)

PuraStat® - Gamma PuraStat® - Gamma

irradiated at 25 KGy Shearing at 1000s-1 irradiated at 23 KGy

PuraStat® - Control

10000

1000

100 10

60 run 2nd after recovery G' 60min 40

20

1/0 K- 1 min-

shear rate = 1000s

Time (min)

60 run 1st after recovery G' 40 60min

20

1/0 1 min- 1 min-

shear rate =

1000s

1/0 Initial G'

Figure 9

<-

10000 1000 100 10 0